血管内皮生长因子转染骨髓间充质干细胞心肌移植对心肌梗死后大鼠心功能及血管新生的作用

目的建立重组人血管内皮生长因子(VEGF)165基因转染大鼠骨髓间充质干细胞(MSC)的方法,探讨该细胞心肌移植对缺血性心脏病心功能及血管新生的影响,并比较联合治疗与单独基因或细胞治疗的疗效差异。方法采用密度梯度离心-贴壁培养法获取Wistar近交系大鼠MSC,用脂质体将pcDNA3.1-hVEGF165转入该细胞,通过ELISA、RT—PCR和Western印迹检测后者VEGF的表达情况;以Wistar近交系大鼠建立心肌缺血模型,随机分成4组(每组12只),心肌梗死模型建立2周后,联合组在心肌梗死区移植转染VEGF165基因的MSC,细胞组移植等量的MSC,基因组注射脂质体-pcDNA3.1-VEGF165DNA复合物,对照组注射等容积培养液;另取12只未结扎冠脉的大鼠为假手术组。细胞移植4周后,用Buxco系统检测心功能;然后处死动物,取心肌标本,测量心肌梗死面积;用5-溴脱氧尿嘧啶(Brdu)、肌钙蛋白T免疫组化双染法确定移植细胞的存活与分化;用Ⅷ因子染色法检测血管新生,RT-PCR法检测VEGF165基因的体内表达情况。结果(1)pcDNA3.1-hVEGF165基因通过脂质体转染大鼠MSC后获稳定表达;(2)移植4周后,联合组心肌梗死面积(27．8％±3．0％)明显低于细胞组(37．0％±10．1％)与基因组(37．1％±5．2％,均P<0．05),心功能改善优于细胞组与基因组;(3)联合组心肌梗死区毛细血管密度(每视野40．2个±5．5个)高于细胞组(27．2个±6．3个,P<0．01)和对照组(18．5个±5．8个,P<0．01),较基因组(35．8个±7．7个)亦有增加的趋势(P=0．189);(4)Brdu、肌钙蛋白T双染示各治疗组心肌梗死区心肌细胞数量不同程度的多于对照组;(5)联合组VEGF基因的体内表达(hVEGFmRNA相对含量0．18±0．04)高于基因组(0．10±0．03,P<0．01)。结论转染VEGF基因的MSC移植可使鼠冠脉结扎造成的心肌梗死面积缩小、心功能明显改善,其疗效优于单独应用基因或细胞治疗,为缺血性心脏病的细胞基因联合治疗提供了理论依据。

Effects of myocardial transplantation of mesenchymal stem cells transfected with vascular endothelial factor gene on improvement of heart function and angiogenesis after myocardial infarction: experiment with rats

To establish a method to transfect vascular endothelial factor (VEGF) gene into mesenchymal stem cells ( MSCs) , to investigate the effects of the gene-transfected MSCs on heart function restoration and angiogenesis after myocardial infarction, and to compare the differences among cell therapy, gene therapy, and combined therapy. METHODS: Seventy-one Wistar rats underwent ligation of the left anterior descending coronary artery so as to establish heart ischemia models. Fifteen rats underwent sham operation. MSCs were isolated from several Wistar rats by density gradient centrifugation, purified, and transfected with pcDNA3.1-hVEGF165 or blank plasmid pcDNA3.1 respectively using the liposome mediated method. ELISA, Western blotting, and RT-PCR were used to detect the protein and mRNA expression of hVEG in these MSCs Forty-eight surviving rats that underwent ligation were randomly divided into 4 equal groups: combination group (Combo group) to be injected into the heart infarct zone with suspension of hVEGF165-transfected MSCs 2 weeks after the establishment of the model, cell group to be injected with suspension of MSCs not transfected with VEGF, gene group to be injected with suspension of DNA-liposome containing pcDNA3.1-VEGF165 and control group to be injected with cold culture fluid only. Twelve surviving rats that underwent sham operation were used as normal non-ischemic group. Four weeks after the injection the surviving rats underwent examination of heart functions by the Buxco system. The rats were killed and their hearts were taken out to undergo immunohistochemistry with 5-bromodeoxyuridine (Brdu) and troponin T and factor VIII to measure the area of cardiac infarction and the capillary density. RT-PCR was used to examine the mRNA expression of VEGF. The heart infarcted size was calculated by Evan's blue staining. RESULTS: (1) MSCs can be successfully isolated and cultured by density gradient centrifugation followed by adherence-separation. The expression of hVEGF165 in the transfected MSCs was demonstrated with ELISA, RT-PCR and Western Blot Assay. (2) Four weeks after the cells were transplanted, among all groups but the nonischemic group, the heart infarcted size of the Combo group was 27.8% +/- 3. 0% ,significantly less than those of the cell group (37.0% +/- 10. 1% ) and gene group (37.1% +/- 5.2%, both P <0.05). The heart function of the Combo group was better than those of other groups. (3) The capillary density of the Combo group was 40. 2 +/- 5.5/visual field, significantly greater than those of both the cell group (27.2 +/- 6. 3/visual field, P <0. 01) and that of the control group (18.5 +/- 5.8/visual field, P <0. 01) , and greater to some degree than that of the gene group (35. 8 +/-7.7/visual field, P =0. 189). (4)The heart infarcted size, heart function and capillary density of the cell and gene groups were similar and were smaller, better and greater than those of the control group. (5) Brdu and troponin T double staining detected a varied increase in the number of surviving cardiomyocytes at the heart infarcted area, some of which were double stain positive. RT-PCR showed mRNA expression of hVEGF165 in the Combo and gene groups, that in the Combo group being higher than that in the gene group. CONCLUSION: Eukaryotic expression vector pcDNA3.1-hVEGF165 can effectively be expressed in MSCs. Transplantation of VEGF gene by means of transfected MSCs brings better improvement in myocardial perfusion and in restoration of heart function than either cellular or gene therapy alone.